BACKGROUND:Tyrosine protein-kinase 2 (TYK2) mediates inflammatory signalling through multiple cytokines, including interferon-α (IFNα), interleukin (IL)-12, and IL-23. TYK2 missense mutations protect against type 1 diabetes (T1D), and inhibition of TYK2 shows promise in other autoimmune conditions. METHODS:We evaluated the effects of specific TYK2 inhibitors (TYK2is) in pre-clinical models of T1D, including human β cells, cadaveric islets, iPSC-derived islets, and mouse models. FINDINGS:In vitro studies showed that TYK2is prevented IFNα-induced β cell HLA class I up-regulation, endoplasmic reticulum stress, and chemokine production. In co-culture studies, pre-treatment of β cells with TYK2i prevented IFNα-induced antigenic peptide presentation and alloreactive and autoreactive T cell degranulation. In vivo administration of BMS-986202 in two mouse models of T1D (RIP-LCMV-GP and NOD mice) reduced systemic and tissue-localised inflammation, prevented β cell death, and delayed T1D onset. Transcriptional phenotyping of pancreatic islets, pancreatic lymph nodes, and spleen highlighted a role for TYK2 inhibition in modulating signalling pathways associated with inflammation, translational control, stress signalling, secretory function, immunity, and diabetes. Additionally, TYK2i treatment changed the composition of innate and adaptive immune cell populations in the blood and disease target tissues. INTERPRETATION:These findings indicate that TYK2i has beneficial effects on both the immune and endocrine compartments in models of T1D, thus supporting a path forward for testing TYK2is in human T1D. FUNDING:This work was supported by the National Institutes of Health (NIH), Veteran Affairs (VA), Breakthrough T1D, and gifts from the Sigma Beta Sorority, the Ball Brothers Foundation, and the George and Frances Ball Foundation.

T cell-based immunotherapies targeting antigens on tumor cells have shown efficacy as anti-cancer treatments. While neoantigens are created by somatic mutations acquired during tumorigenesis, allogeneic stem cell transplantation as treatment for hematological malignancies exploits minor histocompatibility antigens encoded by genetic differences between patients and donors. Screening methods to predict neoantigens and minor histocompatibility antigens typically consider only conventional antigens created by nonsynonymous mutations or polymorphisms coding for amino acid changes in canonical open reading frames (ORFs). However, unconventional ORFs encoding peptides outside the known human proteome also provide an important source of cryptic antigens targeted in anti-tumor responses. Here, we used the recently expanded repertoire of human leukocyte antigen (HLA) class I-restricted minor histocompatibility antigens identified in patients treated with allogeneic stem cell transplantation by method unbiased regarding the type of antigen to explore the sensitivity of ribosome profiling to detect ORFs for different types of T cell antigens. Ribosome profiling showed high sensitivity to detect upstream ORFs for cryptic antigens similar canonical ORFs for conventional antigens, while cryptic antigens in out-of-frame ORFs and ORFs in long non-coding RNAs were largely missed. In conclusion, ribosome profiling shows variable sensitivity to detect ORFs for canonical and different types of cryptic T cell antigens.

Aims/hypothesis The inflammatory milieu characteristic of insulitis affects translation fidelity and generates defective ribosomal products (DRiPs) that participate in autoimmune beta cell destruction in type 1 diabetes. Here, we studied the role of early innate cytokines (IFNα) and late immune adaptive events (IFNɣ) in insulin DRiP-derived peptide presentation to diabetogenic CD8+ T cells. Methods Single-cell transcriptomics of human pancreatic islets was used to study the composition of the (immuno)proteasome. Specific inhibition of the immunoproteasome catalytic subunits was achieved using siRNA, and antigenic peptide presentation at the cell surface of the human beta cell line EndoC-βH1 was monitored using peptide-specific CD8 T cells. Results We found that IFNγ induces the expression of the PSMB10 transcript encoding the β2i catalytic subunit of the immunoproteasome in endocrine beta cells, revealing a critical role in insulin DRiP-derived peptide presentation to T cells. Moreover, we showed that PSMB10 is upregulated in a beta cell subset that is preferentially destroyed in the pancreases of individuals with type 1 diabetes. Conclusions/interpretation Our data highlight the role of the degradation machinery in beta cell immunogenicity and emphasise the need for evaluation of targeted immunoproteasome inhibitors to limit beta cell destruction in type 1 diabetes. Data availability The single-cell RNA-seq dataset is available from the Gene Expression Omnibus (GEO) using the accession number GSE218316 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE218316 ). Graphical Abstract

Transcriptome analyses revealed insulin-gene-derived transcripts in non-beta endocrine islet cells. We studied alternative splicing of human INS mRNA in pancreatic islets. Alternative splicing of insulin pre-mRNA was determined by PCR analysis performed on human islet RNA and single-cell RNA-seq analysis. Antisera were generated to detect insulin variants in human pancreatic tissue using immunohistochemistry, electron microscopy and single-cell western blot to confirm the expression of insulin variants. Cytotoxic T lymphocyte (CTL) activation was determined by MIP-1β release. We identified an alternatively spliced INS product. This variant encodes the complete insulin signal peptide and B chain and an alternative C-terminus that largely overlaps with a previously identified defective ribosomal product of INS. Immunohistochemical analysis revealed that the translation product of this INS-derived splice transcript was detectable in somatostatin-producing delta cells but not in beta cells; this was confirmed by light and electron microscopy. Expression of this alternatively spliced INS product activated preproinsulin-specific CTLs in vitro. The exclusive presence of this alternatively spliced INS product in delta cells may be explained by its clearance from beta cells by insulin-degrading enzyme capturing its insulin B chain fragment and a lack of insulin-degrading enzyme expression in delta cells. Our data demonstrate that delta cells can express an INS product derived from alternative splicing, containing both the diabetogenic insulin signal peptide and B chain, in their secretory granules. We propose that this alternative INS product may play a role in islet autoimmunity and pathology, as well as endocrine or paracrine function or islet development and endocrine destiny, and transdifferentiation between endocrine cells. INS promoter activity is not confined to beta cells and should be used with care when assigning beta cell identity and selectivity. The full EM dataset is available via www.nanotomy.org (for review: http://www.nanotomy.org/OA/Tienhoven2021SUB/6126-368/ ). Single-cell RNA-seq data was made available by Segerstolpe et al [13] and can be found at https://sandberglab.se/pancreas . The RNA and protein sequence of INS-splice was uploaded to GenBank (BankIt2546444 INS-splice OM489474).

OBJECTIVES:Readily accessible human pancreatic beta cells that are functionally close to primary adult beta cells are a crucial model to better understand human beta cell physiology and develop new treatments for diabetes. We here report the characterization of EndoC-βH5 cells, the latest in the EndoC-βH cell family.METHODS:EndoC-βH5 cells were generated by integrative gene transfer of immortalizing transgenes hTERT and SV40 large T along with Herpes Simplex Virus-1 thymidine kinase into human fetal pancreas. Immortalizing transgenes were removed after amplification using CRE activation and remaining non-excized cells eliminated using ganciclovir. Resulting cells were distributed as ready to use EndoC-βH5 cells. We performed transcriptome, immunological and extensive functional assays.RESULTS:Ready to use EndoC-βH5 cells display highly efficient glucose dependent insulin secretion. A robust 10-fold insulin secretion index was observed and reproduced in four independent laboratories across Europe. EndoC-βH5 cells secrete insulin in a dynamic manner in response to glucose and secretion is further potentiated by GIP and GLP-1 analogs. RNA-seq confirmed abundant expression of beta cell transcription factors and functional markers, including incretin receptors. Cytokines induce a gene expression signature of inflammatory pathways and antigen processing and presentation. Finally, modified HLA-A2 expressing EndoC-βH5 cells elicit specific A2-alloreactive CD8 T cell activation.CONCLUSIONS:EndoC-βH5 cells represent a unique storable and ready to use human pancreatic beta cell model with highly robust and reproducible features. Such cells are thus relevant for the study of beta cell function, screening and validation of new drugs, and development of disease models.

Interferon-α (IFNα) plays a prominent role in type 1 diabetes (T1D) pathogenesis and mediates its effects through the IFN receptor (IFNAR) and the protein tyrosine kinases JAK1 and TYK2. Polymorphisms that decrease TYK2 activity are protective against T1D, and TYK2 inhibitors (TYKi) are being evaluated for therapeutic benefit in other autoimmune conditions. To test whether TYK2 inhibitors BMS-986202 and BMS-986165 have similar efficacy in diverse models of T1D, we evaluated their effect in vitro on human islets, EndoC-βH1 cells, and iPSC-derived islet-like aggregates and monitored diabetes incidence in RIP-LCMV and NOD mice following in vivo treatment with BMS-986202. TYK2i prevented IFNα-mediated upregulation of CXCL10, MX1, and HLA-ABC in human islets, iPSCs, and EndoC-βH1 and decreased IFNα-mediated STAT1/2 phosphorylation and apoptosis in human islets. Importantly, BMS-986202 reduced diabetes incidence in RIP-LCMV mice by 80% (n=18 vehicle/18 TYK2i; p<0.001). Flow cytometry analysis 3 days post LCMV injection revealed a decrease in the percentage of CD11b+F4/80+ macrophages (M1) and CD11b+CD49+ NK cells in pancreatic lymph nodes (PLN) and blood and an increase in circulating CD11b-CD49+ tolerogenic-NK cells in TYK2i-treated RIP-LCMV mice. At days 7 and 14 post LCMV, PDI+CD8+T-cells in the blood, spleen, and PLN and PDI+FOXP3+ Treg cells in the spleen were increased in TYK2i-treated RIP-LCMV mice, indicating T-cell exhaustion. Similarly, treatment with BMS-986202 resulted in a 44% decrease in diabetes incidence in NOD mice (n=32 vehicle/34 TYK2i; p=0.0075) and a significant reduction in insulitis (p<0.05). smFISH analysis revealed decreased β cell expression of STAT1 and MX1 in TYK2i-treated RIP-LCMV mice and NOD mice compared to vehicle-treated mice, while T-cell activation upon co-incubation with target human β cells was inhibited by TYK2i treatment. Taken together, our findings demonstrate that TYK2 inhibition protects against β cell inflammation and T1D development in multiple preclinical models. Disclosure F.Syed: None. M.I.Alvelos: None. G.Chang: None. K.Orr: None. K.Yamada: None. J.Liu: None. A.Zaldumbide: None. D.Scheuner: None. D.L.Eizirik: None. C.Evans-molina: Advisory Panel; Provention Bio, Inc., DiogenX, Avotres Inc., Neurodon, MaiCell Therapeutics, Other Relationship; Isla Technology, Bristol-Myers Squibb Company, Nimbus Therapeutics, Research Support; Lilly, Astellas Pharma Inc. O.Ballew: None. C.Lee: None. J.Rana: Research Support; Bayer Inc. A.Castela: None. S.A.Weaver: Employee; Eli Lilly and Company. S.Thomaidou: None. S.Demine: None. A.Coomans de brachène: None. Funding National Institutes of Health (DK127308, UC4DK104166); JDRF (2-SRA-2019-834-S-B, 5-CDA-2022-1176-A-N)

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

IFNα is a key regulator of the dialogue between pancreatic β cells and the immune system in early type 1 diabetes (T1D). IFNα up-regulates HLA class I expression in human β cells, fostering autoantigen presentation to the immune system. We observed by bulk and single-cell RNA sequencing that exposure of human induced pluripotent-derived islet-like cells to IFNα induces expression of HLA class I and of other genes involved in antigen presentation, including the transcriptional activator NLRC5. We next evaluated the global role of NLRC5 in human insulin-producing EndoC-βH1 and human islet cells by RNA sequencing and targeted gene/protein determination. NLRC5 regulates expression of HLA class I, antigen presentation–related genes, and chemokines. NLRC5 also mediates the effects of IFNα on alternative splicing, a generator of β cell neoantigens, suggesting that it is a central player of the effects of IFNα on β cells that contribute to trigger and amplify autoimmunity in T1D.

Introduction Enterovirus infection has long been suspected as a possible trigger for type 1 diabetes. Upon infection, viral double-stranded RNA (dsRNA) is recognized by membrane and cytosolic sensors that orchestrate type I interferon signaling and the recruitment of innate immune cells to the pancreatic islets. In this context, adenosine deaminase acting on RNA 1 (ADAR1) editing plays an important role in dampening the immune response by inducing adenosine mispairing, destabilizing the RNA duplexes and thus preventing excessive immune activation. Methods Using high-throughput RNA sequencing data from human islets and EndoC-βH1 cells exposed to IFNα or IFNγ/IL1β, we evaluated the role of ADAR1 in human pancreatic β cells and determined the impact of the type 1 diabetes pathophysiological environment on ADAR1-dependent RNA editing. Results We show that both IFNα and IFNγ/IL1β stimulation promote ADAR1 expression and increase the A-to-I RNA editing of Alu-Containing mRNAs in EndoC-βH1 cells as well as in primary human islets. Discussion We demonstrate that ADAR1 overexpression inhibits type I interferon response signaling, while ADAR1 silencing potentiates IFNα effects. In addition, ADAR1 overexpression triggers the generation of alternatively spliced mRNAs, highlighting a novel role for ADAR1 as a regulator of the β cell transcriptome under inflammatory conditions.

Type 1 diabetes is an autoimmune disease characterized by autoreactiveT-cell mediated destruction of the insulin-producing pancreatic beta-cells.Increasing evidence suggest that the beta-cells themselves contribute to theirown destruction by generating neo-antigens through the production of aberrantor modified proteins that escape central tolerance. We have recentlydemonstrated that ribosomal infidelity amplified by stress could lead to thegeneration of neoantigens in human beta-cells, emphasizing the participation ofnonconventional translation events to autoimmunity, as occurring in cancer orvirus-infected tissues. Using a transcriptome-wide profiling approach to maptranslation initiation start sites in human beta-cells under standard andinflammatory conditions, we identify a completely new set of polypeptidesderived from non-canonical start sites and translation initiation withinlncRNA. Our data underline the extreme diversity of the beta-cell translatomeand may reveal new functional biomarkers for beta-cell distress, diseaseprediction and progression and therapeutic intervention in type 1 diabetes.

The signal peptide of preproinsulin is a major source for HLA class I autoantigen epitopes implicated in CD8 T cell (CTL)–mediated β-cell destruction in type 1 diabetes (T1D). Among them, the 10-mer epitope located at the C-terminal end of the signal peptide was found to be the most prevalent in patients with recent-onset T1D. While the combined action of signal peptide peptidase and endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) is required for processing of the signal peptide, the mechanisms controlling signal peptide trimming and the contribution of the T1D inflammatory milieu on these mechanisms are unknown. Here, we show in human β-cells that ER stress regulates ERAP1 gene expression at posttranscriptional level via the IRE1α/miR-17-5p axis and demonstrate that inhibition of the IRE1α activity impairs processing of preproinsulin signal peptide antigen and its recognition by specific autoreactive CTLs during inflammation. These results underscore the impact of ER stress in the increased visibility of β-cells to the immune system and position the IRE1α/miR-17 pathway as a central component in β-cell destruction processes and as a potential target for the treatment of autoimmune T1D.

Type 1 diabetes mellitus is believed to result from destruction of the insulin-producing β-cells in pancreatic islets that is mediated by autoimmune mechanisms. The classic view is that autoreactive T cells mistakenly destroy healthy (‘innocent’) β-cells. We propose an alternative view in which the β-cell is the key contributor to the disease. By their nature and function, β-cells are prone to biosynthetic stress with limited measures for self-defence. β-Cell stress provokes an immune attack that has considerable negative effects on the source of a vital hormone. This view would explain why immunotherapy at best delays progression of type 1 diabetes mellitus and points to opportunities to use therapies that revitalize β-cells, in combination with immune intervention strategies, to reverse the disease. We present the case that dysfunction occurs in both the immune system and β-cells, which provokes further dysfunction, and present the evidence leading to the consensus that islet autoimmunity is an essential component in the pathogenesis of type 1 diabetes mellitus. Next, we build the case for the β-cell as the trigger of an autoimmune response, supported by analogies in cancer and antitumour immunity. Finally, we synthesize a model (‘connecting the dots’) in which both β-cell stress and islet autoimmunity can be harnessed as targets for intervention strategies.

β‐cell destruction in type 1 diabetes (T1D) results from the effect of inflammation and autoimmunity. In response to inflammatory signals, islet cells engage adaptive mechanisms to restore and maintain cellular homeostasis. Among these mechanisms, the unfolded protein response (UPR) leads to a reduction of the general protein translation rate, increased production of endoplasmic reticulum chaperones and the initiation of degradation by activation of the ER associated degradation pathway (ERAD) in which newly synthetized proteins are ubiquitinylated and processed through the proteasome. This adaptive phase is also believed to play a critical role in the development of autoimmunity by the generation of neoantigens. While we have previously investigated the effect of stress on transcription, translation and post‐translational events as possible source for neoantigens, the participation of the degradation machinery, yet crucial in the generation of antigenic peptides, remains to be investigated in the context of T1D pathology. In this review, we will describe the relation between the unfolded protein response and the Ubiquitin Proteasome System (UPS) and address the role of the cellular degradation machinery in the generation of antigens. Learning from tumour immunology, we propose how these processes may unmask β‐cells by triggering the generation of aberrant peptides recognized by the immune cells.